Molecular mechanism of magnesium alloy promoting macrophage M2 polarization through modulation of PI3K/AKT signaling pathway for tendon bone healing in rotator cuff injury repair_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

SHENG Xianhao ^1,2 , ZHANG Wen ³ , SONG Shoulong ^1,2 , ZHANG Fei ^1,2 , ZHANG Baoxiang ³ , TIAN Xiaoying ³ , XIONG Wentao ^1,2 , ZHU Yingguang ^1,2 , XIE Yuxin ^1,2 , LI Zi’ang ^1,2 , TAN Lili ³ , ZHANG Qiang ² ,  WANG Yan ²

1. Chinese PLA Medical School, Beijing, 100853, P. R. China;
2. Department of Orthopedics, the Fourth Medical Center of Chinese PLA General Hospital, Beijing, 100048, P. R. China;
3. Institute of Metal Research, Chinese Academy of Sciences, Shenyang Liaoning, 110013, P. R. China;

Corresponding author：

ZHANG Qiang, Email: ; WANG Yan, Email: yanwang301@163.com

Keywords：

Magnesium alloy; rotator cuff tear; tendon-bone healing; phosphatidylinositol 3 kinase; protein kinase B; signaling pathway; macrophage

DOI：

10.7507/1002-1892.202410010

Video：

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Objective To evaluate the effectiveness of biodegradable magnesium alloy materials in promoting tendon-bone healing during rotator cuff tear repair and to investigate their potential underlying biological mechanisms. Methods Forty-eight 8-week-old Sprague Dawley rats were taken and randomly divided into groups A, B, and C. Rotator cuff tear models were created and repaired using magnesium alloy sutures in group A and Vicryl Plus 4-0 absorbable sutures in group B, while only subcutaneous incisions and sutures were performed in group C. Organ samples of groups A and B were taken for HE staining at 1 and 2 weeks after operation to evaluate the safety of magnesium alloy, and specimens from the supraspinatus tendon and proximal humerus were harvested at 2, 4, 8, and 12 weeks after operation. The specimens were observed macroscopically at 4 and 12 weeks after operation. Biomechanical tests were performed at 4, 8, and 12 weeks to test the ultimate load and stiffness of the healing sites in groups A and B. At 2, 4, and 12 weeks, the specimens were subjected to the following tests: Micro-CT to evaluate the formation of bone tunnels in groups A and B, HE staining and Masson staining to observe the regeneration of fibrocartilage at the tendon-bone interface after decalcification and sectioning, and Goldner trichrome staining to evaluate the calcification. Immunohistochemical staining was performed to detect the expression of angiogenic factors, including vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2), as well as osteogenic factors at the tendon-bone interface. Additionally, immunofluorescence staining was used to examine the expression of arginase 1 and Integrin beta-2 to assess M1 and M2 macrophage polarization at the tendon-bone interface. The role of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in tendon-bone healing was further analyzed using real-time fluorescence quantitative PCR. Results Analysis of visceral sections revealed that magnesium ions released during the degradation of magnesium alloys did not cause significant toxic effects on internal organs such as the heart, liver, spleen, lungs, and kidneys, indicating good biosafety. Histological analysis further demonstrated that fibrocartilage regeneration at the tendon-bone interface in group A occurred earlier, and the amount of fibrocartilage was significantly greater compared to group B, suggesting a positive effect of magnesium alloy material on tendon-bone interface repair. Additionally, Micro-CT analysis revealed that bone tunnel formation occurred more rapidly in group A compared to group B, further supporting the beneficial effect of magnesium alloy on bone healing. Biomechanical testing showed that the ultimate loads in group A were consistently higher than in group B, at 4 weeks, the stiffness of group A was also greater than that of group B, indicating stronger tissue-carrying capacity following tendon-bone interface repair and highlighting the potential of magnesium alloy in enhancing tendon-bone healing. Immunohistochemical staining results indicated that the expression of VEGF and BMP-2 was significantly upregulated during the early stages of healing, suggesting that magnesium alloy effectively promoted angiogenesis and bone formation, thereby accelerating the tendon-bone healing process. Immunofluorescence staining further revealed that magnesium ions exerted significant anti-inflammatory effects by regulating macrophage polarization, promoting their shift toward the M2 phenotype. Real-time fluorescence quantitative PCR results demonstrated that magnesium ions could facilitate tendon-bone healing by modulating the PI3K/AKT signaling pathway, which represented one of the molecular mechanisms driving this healing process. Conclusion Biodegradable magnesium alloy material accelerated fibrocartilage regeneration and calcification at the tendon-bone interface in rat rotator cuff tear repair by regulating the PI3K/AKT signaling pathway, thereby significantly enhancing tendon-bone healing.

1.	Longo UG, Carnevale A, Piergentili I, et al. Retear rates after rotator cuff surgery: a systematic review and meta-analysis. BMC Musculoskelet Disord, 2021, 22(1): 749. doi: 10.1186/s12891-021-04634-6.
2.	Villarreal-Villarreal GA, Simental-Mendía M, Garza-Borjón AE, et al. Double-row rotator cuff repair enhanced with platelet-rich therapy reduces retear rate: A systematic review and meta-analysis of randomized controlled trials. Arthroscopy, 2021, 37(6): 1937-1947.
3.	Bedi A, Bishop J, Keener J, et al. Rotator cuff tears. Nat Rev Dis Primers, 2024, 10(1): 8. doi: 10.1038/s41572-024-00492-3.
4.	Heilig P, Jordan MC, Paul MM, et al. Augmentation of suture anchors with magnesium phosphate cement - Simple technique with striking effect. J Mech Behav Biomed Mater, 2022, 128: 105096. doi: 10.1016/j.jmbbm.2022.105096.
5.	Cobaleda Aristizabal AF, Sanders EJ, Barber FA. Adverse events associated with biodegradable lactide-containing suture anchors. Arthroscopy, 2014, 30(5): 555-560.
6.	吴雨宽, 白浪, 刘妍兰, 等. 镁及镁合金植入物在运动医学中的应用研究进展. 中国修复重建外科杂志, 2024, 38(3): 380-386.
7.	Karunakaran R, Ortgies S, Tamayol A, et al. Additive manufacturing of magnesium alloys. Bioact Mater, 2020, 5(1): 44-54.
8.	Puccetti M, Cusati E, Antognelli C, et al. Ketorolac loaded poly(lactic-co-glycolic acid) coating of AZ31 in the treatment of bone fracture pain. Polymers (Basel), 2023, 15(10): 2246. doi: 10.3390/polym15102246.
9.	Zhang B, Zhang W, Zhang F, et al. Degradable magnesium alloy suture promotes fibrocartilaginous interface regeneration in a rat rotator cuff transosseous repair model. Journal of Magnesium and Alloys, 2024, 12(1): 384-393.
10.	He X, Li Y, Zou D, et al. An overview of magnesium-based implants in orthopaedics and a prospect of its application in spine fusion. Bioact Mater, 2024, 39: 456-478.
11.	Zhang W, Sheng X, Zhang B, et al. A novel design magnesium alloy suture anchor promotes fibrocartilaginous enthesis regeneration in rabbit rotator cuff repair. Journal of Magnesium and Alloys, 2024. doi:10.1016/j.jma.2024.08.002.
12.	Iwahashi T, Shino K, Nakata K, et al. Direct anterior cruciate ligament insertion to the femur assessed by histology and 3-dimensional volume-rendered computed tomography. Arthroscopy, 2010, 26(9 Suppl): S13-S20.
13.	Zhang X, Ma J, Hu H, et al. Engineered metallic ion-based hydrogel for tendon-bone reconstruction. ACS Appl Mater Interfaces, 2024, 16(6): 6837-6848.
14.	Bai L, Kasimu A, Wang S, et al. Electrohydrodynamic-printed dual-triphase microfibrous scaffolds reshaping the lipidomic profile for enthesis healing in a rat rotator cuff repair model. Small, 2024. doi: 10.1002/smll.202406069.
15.	Zhao Q, Ni Y, Wei H, et al. Ion incorporation into bone grafting materials. Periodontol 2000, 2024, 94(1): 213-230.
16.	Zhang E, Zhao X, Hu J, et al. Antibacterial metals and alloys for potential biomedical implants. Bioact Mater, 2021, 6(8): 2569-2612.
17.	Canales DA, Reyes F, Saavedra M, et al. Electrospun fibers of poly (lactic acid) containing bioactive glass and magnesium oxide nanoparticles for bone tissue regeneration. Int J Biol Macromol, 2022, 210: 324-336.
18.	Nan M, Yangmei C, Bangcheng Y. Magnesium metal—a potential biomaterial with antibone cancer properties. J Biomed Mater Res A, 2014, 102(8): 2644-2651.
19.	He X, Li Y, Miao H, et al. High formability Mg-Zn-Gd wire facilitates ACL reconstruction via its swift degradation to accelerate intra-tunnel endochondral ossification. Journal of Magnesium and Alloys, 2024, 12(1): 295-315.
20.	Chen F, Liang Q, Mao L, et al. Synergy effects of Asperosaponin Ⅵ and bioactive factor BMP-2 on osteogenesis and anti-osteoclastogenesis. Bioact Mater, 2021, 10: 335-344.
21.	Pugliese E, Sallent I, Ribeiro S, et al. Development of three-layer collagen scaffolds to spatially direct tissue-specific cell differentiation for enthesis repair. Mater Today Bio, 2023, 19: 100584. doi: 10.1016/j.mtbio.2023.100584.
22.	Ding T, Kang W, Li J, et al. An in situ tissue engineering scaffold with growth factors combining angiogenesis and osteoimmunomodulatory functions for advanced periodontal bone regeneration. J Nanobiotechnology, 2021, 19(1): 247. doi: 10.1186/s12951-021-00992-4.
23.	Hashimoto Y, Yoshida G, Toyoda H, et al. Generation of tendon-to-bone interface “enthesis” with use of recombinant BMP-2 in a rabbit model. J Orthop Res, 2007, 25(11): 1415-1424.
24.	Kim JG, Kim HJ, Kim SE, et al. Enhancement of tendon-bone healing with the use of bone morphogenetic protein-2 inserted into the suture anchor hole in a rabbit patellar tendon model. Cytotherapy, 2014, 16(6): 857-867.
25.	Rodeo SA. Biologic augmentation of rotator cuff tendon repair. J Shoulder Elbow Surg, 2007, 16(5 Suppl): S191-S197.
26.	杜志坡, 廖婕, 王冰冰, 等. 细胞来源脱细胞外基质用作组织工程支架的优势与展望. 中国修复重建外科杂志, 2024, 38(11): 1291-1298.
27.	Li J, Ke H, Lei X, et al. Controlled-release hydrogel loaded with magnesium-based nanoflowers synergize immunomodulation and cartilage regeneration in tendon-bone healing. Bioact Mater, 2024, 36: 62-82.
28.	Cai Z, Liu X, Hu M, et al. In situ enzymatic reaction generates magnesium-based mineralized microspheres with superior bioactivity for enhanced bone regeneration. Adv Healthc Mater, 2023, 12(24): e2300727. doi: 10.1002/adhm.202300727.
29.	Sun Y, Liu H, Sun XY, et al. In vitro and in vivo study on the osseointegration of magnesium and strontium ion with two different proportions of mineralized collagen and its mechanism. J Biomater Appl, 2021, 36(3): 528-540.
30.	Zhang X, Zu H, Zhao D, et al. Ion channel functional protein kinase TRPM7 regulates Mg ions to promote the osteoinduction of human osteoblast via PI3K pathway: In vitro simulation of the bone-repairing effect of Mg-based alloy implant. Acta Biomater, 2017, 63: 369-382.
31.	Wang Z, Wang X, Pei J, et al. Degradation and osteogenic induction of a SrHPO4-coated Mg-Nd-Zn-Zr alloy intramedullary nail in a rat femoral shaft fracture model. Biomaterials, 2020, 247: 119962. doi: 10.1016/j.biomaterials.2022.121375.
32.	Zhao M, Dai Y, Li X, et al. Evaluation of long-term biocompatibility and osteogenic differentiation of graphene nanosheet doped calcium phosphate-chitosan AZ91D composites. Mater Sci Eng C Mater Biol Appl, 2018, 90: 365-378.
33.	Cheng P, Weng Z, Hamushan M, et al. High-purity magnesium screws modulate macrophage polarization during the tendon-bone healing process in the anterior cruciate ligament reconstruction rabbit model. Regen Biomater, 2022, 9: rbac067. doi: 10.1093/rb/rbac067.
34.	Wang F, Sun P, Xie E, et al. Phytic acid/magnesium ion complex coating on PEEK fiber woven fabric as an artificial ligament with anti-fibrogenesis and osteogenesis for ligament-bone healing. Biomater Adv, 2022, 140: 213079. doi: 10.1016/j.bioadv.2022.213079.

1. Longo UG, Carnevale A, Piergentili I, et al. Retear rates after rotator cuff surgery: a systematic review and meta-analysis. BMC Musculoskelet Disord, 2021, 22(1): 749. doi: 10.1186/s12891-021-04634-6.
2. Villarreal-Villarreal GA, Simental-Mendía M, Garza-Borjón AE, et al. Double-row rotator cuff repair enhanced with platelet-rich therapy reduces retear rate: A systematic review and meta-analysis of randomized controlled trials. Arthroscopy, 2021, 37(6): 1937-1947.
3. Bedi A, Bishop J, Keener J, et al. Rotator cuff tears. Nat Rev Dis Primers, 2024, 10(1): 8. doi: 10.1038/s41572-024-00492-3.
4. Heilig P, Jordan MC, Paul MM, et al. Augmentation of suture anchors with magnesium phosphate cement - Simple technique with striking effect. J Mech Behav Biomed Mater, 2022, 128: 105096. doi: 10.1016/j.jmbbm.2022.105096.
5. Cobaleda Aristizabal AF, Sanders EJ, Barber FA. Adverse events associated with biodegradable lactide-containing suture anchors. Arthroscopy, 2014, 30(5): 555-560.
6. 吴雨宽, 白浪, 刘妍兰, 等. 镁及镁合金植入物在运动医学中的应用研究进展. 中国修复重建外科杂志, 2024, 38(3): 380-386.
7. Karunakaran R, Ortgies S, Tamayol A, et al. Additive manufacturing of magnesium alloys. Bioact Mater, 2020, 5(1): 44-54.
8. Puccetti M, Cusati E, Antognelli C, et al. Ketorolac loaded poly(lactic-co-glycolic acid) coating of AZ31 in the treatment of bone fracture pain. Polymers (Basel), 2023, 15(10): 2246. doi: 10.3390/polym15102246.
9. Zhang B, Zhang W, Zhang F, et al. Degradable magnesium alloy suture promotes fibrocartilaginous interface regeneration in a rat rotator cuff transosseous repair model. Journal of Magnesium and Alloys, 2024, 12(1): 384-393.
10. He X, Li Y, Zou D, et al. An overview of magnesium-based implants in orthopaedics and a prospect of its application in spine fusion. Bioact Mater, 2024, 39: 456-478.
11. Zhang W, Sheng X, Zhang B, et al. A novel design magnesium alloy suture anchor promotes fibrocartilaginous enthesis regeneration in rabbit rotator cuff repair. Journal of Magnesium and Alloys, 2024. doi:10.1016/j.jma.2024.08.002.
12. Iwahashi T, Shino K, Nakata K, et al. Direct anterior cruciate ligament insertion to the femur assessed by histology and 3-dimensional volume-rendered computed tomography. Arthroscopy, 2010, 26(9 Suppl): S13-S20.
13. Zhang X, Ma J, Hu H, et al. Engineered metallic ion-based hydrogel for tendon-bone reconstruction. ACS Appl Mater Interfaces, 2024, 16(6): 6837-6848.
14. Bai L, Kasimu A, Wang S, et al. Electrohydrodynamic-printed dual-triphase microfibrous scaffolds reshaping the lipidomic profile for enthesis healing in a rat rotator cuff repair model. Small, 2024. doi: 10.1002/smll.202406069.
15. Zhao Q, Ni Y, Wei H, et al. Ion incorporation into bone grafting materials. Periodontol 2000, 2024, 94(1): 213-230.
16. Zhang E, Zhao X, Hu J, et al. Antibacterial metals and alloys for potential biomedical implants. Bioact Mater, 2021, 6(8): 2569-2612.
17. Canales DA, Reyes F, Saavedra M, et al. Electrospun fibers of poly (lactic acid) containing bioactive glass and magnesium oxide nanoparticles for bone tissue regeneration. Int J Biol Macromol, 2022, 210: 324-336.
18. Nan M, Yangmei C, Bangcheng Y. Magnesium metal—a potential biomaterial with antibone cancer properties. J Biomed Mater Res A, 2014, 102(8): 2644-2651.
19. He X, Li Y, Miao H, et al. High formability Mg-Zn-Gd wire facilitates ACL reconstruction via its swift degradation to accelerate intra-tunnel endochondral ossification. Journal of Magnesium and Alloys, 2024, 12(1): 295-315.
20. Chen F, Liang Q, Mao L, et al. Synergy effects of Asperosaponin Ⅵ and bioactive factor BMP-2 on osteogenesis and anti-osteoclastogenesis. Bioact Mater, 2021, 10: 335-344.
21. Pugliese E, Sallent I, Ribeiro S, et al. Development of three-layer collagen scaffolds to spatially direct tissue-specific cell differentiation for enthesis repair. Mater Today Bio, 2023, 19: 100584. doi: 10.1016/j.mtbio.2023.100584.
22. Ding T, Kang W, Li J, et al. An in situ tissue engineering scaffold with growth factors combining angiogenesis and osteoimmunomodulatory functions for advanced periodontal bone regeneration. J Nanobiotechnology, 2021, 19(1): 247. doi: 10.1186/s12951-021-00992-4.
23. Hashimoto Y, Yoshida G, Toyoda H, et al. Generation of tendon-to-bone interface “enthesis” with use of recombinant BMP-2 in a rabbit model. J Orthop Res, 2007, 25(11): 1415-1424.
24. Kim JG, Kim HJ, Kim SE, et al. Enhancement of tendon-bone healing with the use of bone morphogenetic protein-2 inserted into the suture anchor hole in a rabbit patellar tendon model. Cytotherapy, 2014, 16(6): 857-867.
25. Rodeo SA. Biologic augmentation of rotator cuff tendon repair. J Shoulder Elbow Surg, 2007, 16(5 Suppl): S191-S197.
26. 杜志坡, 廖婕, 王冰冰, 等. 细胞来源脱细胞外基质用作组织工程支架的优势与展望. 中国修复重建外科杂志, 2024, 38(11): 1291-1298.
27. Li J, Ke H, Lei X, et al. Controlled-release hydrogel loaded with magnesium-based nanoflowers synergize immunomodulation and cartilage regeneration in tendon-bone healing. Bioact Mater, 2024, 36: 62-82.
28. Cai Z, Liu X, Hu M, et al. In situ enzymatic reaction generates magnesium-based mineralized microspheres with superior bioactivity for enhanced bone regeneration. Adv Healthc Mater, 2023, 12(24): e2300727. doi: 10.1002/adhm.202300727.
29. Sun Y, Liu H, Sun XY, et al. In vitro and in vivo study on the osseointegration of magnesium and strontium ion with two different proportions of mineralized collagen and its mechanism. J Biomater Appl, 2021, 36(3): 528-540.
30. Zhang X, Zu H, Zhao D, et al. Ion channel functional protein kinase TRPM7 regulates Mg ions to promote the osteoinduction of human osteoblast via PI3K pathway: In vitro simulation of the bone-repairing effect of Mg-based alloy implant. Acta Biomater, 2017, 63: 369-382.
31. Wang Z, Wang X, Pei J, et al. Degradation and osteogenic induction of a SrHPO4-coated Mg-Nd-Zn-Zr alloy intramedullary nail in a rat femoral shaft fracture model. Biomaterials, 2020, 247: 119962. doi: 10.1016/j.biomaterials.2022.121375.
32. Zhao M, Dai Y, Li X, et al. Evaluation of long-term biocompatibility and osteogenic differentiation of graphene nanosheet doped calcium phosphate-chitosan AZ91D composites. Mater Sci Eng C Mater Biol Appl, 2018, 90: 365-378.
33. Cheng P, Weng Z, Hamushan M, et al. High-purity magnesium screws modulate macrophage polarization during the tendon-bone healing process in the anterior cruciate ligament reconstruction rabbit model. Regen Biomater, 2022, 9: rbac067. doi: 10.1093/rb/rbac067.
34. Wang F, Sun P, Xie E, et al. Phytic acid/magnesium ion complex coating on PEEK fiber woven fabric as an artificial ligament with anti-fibrogenesis and osteogenesis for ligament-bone healing. Biomater Adv, 2022, 140: 213079. doi: 10.1016/j.bioadv.2022.213079.

Chinese Journal of Reparative and Reconstructive Surgery

Latest ArticlesMolecular mechanism of magnesium alloy promoting macrophage M2 polarization through modulation of PI3K/AKT signaling pathway for tendon bone healing in rotator cuff injury repair

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